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Analysis of the signaling activities of localization mutants of beta-catenin during axis specification in Xenopus.

Miller JR, Moon RT - J. Cell Biol. (1997)

Bottom Line: Given this unexpected result, we focused on the membrane-tethered form of beta-catenin to resolve the apparent discrepancy between its membrane localization and the hypothesized role of nuclear beta-catenin in establishing dorsal cell fate.Compared with nonphosphorylated beta-catenin, beta-catenin phosphorylated by glycogen synthase kinase-3 preferentially associates with microsomal fractions expressing the cytoplasmic region of N-cadherin.These results suggest that protein-protein interactions of beta-catenin can be influenced by its state of phosphorylation, in addition to prior evidence that this phosphorylation modulates the stability of beta-catenin.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, University of Washington School of Medicine, Seattle 98195, USA.

ABSTRACT
In Xenopus embryos, beta-catenin has been shown to be both necessary and sufficient for the establishment of dorsal cell fates. This signaling activity is thought to depend on the binding of beta-catenin to members of the Lef/Tcf family of transcription factors and the regulation of gene expression by this complex. To test whether beta-catenin must accumulate in nuclei to establish dorsal cell fate, we constructed various localization mutants that restrict beta-catenin to either the plasma membrane, the cytosol, or the nucleus. When overexpressed in Xenopus embryos, the proteins localize as predicted, but surprisingly all forms induce an ectopic axis, indicative of inducing dorsal cell fates. Given this unexpected result, we focused on the membrane-tethered form of beta-catenin to resolve the apparent discrepancy between its membrane localization and the hypothesized role of nuclear beta-catenin in establishing dorsal cell fate. We demonstrate that overexpression of membrane-tethered beta-catenin elevates the level of free endogenous beta-catenin, which subsequently accumulates in nuclei. Consistent with the hypothesis that it is this pool of non-membrane-associated beta-catenin that signals in the presence of membrane-tethered beta-catenin, overexpression of cadherin, which binds free beta-catenin, blocks the axis-inducing activity of membrane- tethered beta-catenin. The mechanism by which ectopic membrane-tethered beta-catenin increases the level of endogenous beta-catenin likely involves competition for the adenomatous polyposis coli (APC) protein, which in other systems has been shown to play a role in degradation of beta-catenin. Consistent with this hypothesis, membrane-tethered beta-catenin coimmunoprecipitates with APC and relocalizes APC to the membrane in cells. Similar results are observed with ectopic plakoglobin, casting doubt on a normal role for plakoglobin in axis specification and indicating that ectopic proteins that interact with APC can artifactually elevate the level of endogenous beta-catenin, likely by interfering with its degradation. These results highlight the difficulty in interpreting the activity of an ectopic protein when it is assayed in a background containing the endogenous protein. We next investigated whether the ability of beta-catenin to interact with potential protein partners in the cell may normally be regulated by phosphorylation. Compared with nonphosphorylated beta-catenin, beta-catenin phosphorylated by glycogen synthase kinase-3 preferentially associates with microsomal fractions expressing the cytoplasmic region of N-cadherin. These results suggest that protein-protein interactions of beta-catenin can be influenced by its state of phosphorylation, in addition to prior evidence that this phosphorylation modulates the stability of beta-catenin.

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Overexpression of TM–β-catenin results in the accumulation of endogenous β-catenin in the nucleus. RNA encoding TM– β-catenin 1–9 (A–F) was coinjected with Oregon green dextran (OGDx; B, E, and H) as a lineage tracer to determine the effect of TM– β-catenin overexpression on the distribution of endogenous β-catenin (A, C, D, and F). Merged images (C and F) demonstrate that cells  overexpressing TM–β-catenin 1–9 possess high levels of endogenous β-catenin in nuclei (arrowheads) in contrast to that observed in  nonexpressing cells (arrows). Overexpression of ΔN-9 β-catenin (G–I), that does not promote axis duplication, does not affect the levels  of endogenous β-catenin in the nucleus (G and I; arrowheads mark nuclei of cells expressing the ΔN-9 mutant and arrows mark nuclei of  nonexpressing cells).
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Figure 4: Overexpression of TM–β-catenin results in the accumulation of endogenous β-catenin in the nucleus. RNA encoding TM– β-catenin 1–9 (A–F) was coinjected with Oregon green dextran (OGDx; B, E, and H) as a lineage tracer to determine the effect of TM– β-catenin overexpression on the distribution of endogenous β-catenin (A, C, D, and F). Merged images (C and F) demonstrate that cells overexpressing TM–β-catenin 1–9 possess high levels of endogenous β-catenin in nuclei (arrowheads) in contrast to that observed in nonexpressing cells (arrows). Overexpression of ΔN-9 β-catenin (G–I), that does not promote axis duplication, does not affect the levels of endogenous β-catenin in the nucleus (G and I; arrowheads mark nuclei of cells expressing the ΔN-9 mutant and arrows mark nuclei of nonexpressing cells).

Mentions: Given that the TM–β-catenin mutant appears to signal by elevating a free, signaling pool of endogenous β-catenin, we sought to determine whether overexpression of TM– β-catenin results in the accumulation of endogenous β-catenin in nuclei in a manner similar to that seen after inhibition of GSK-3 (Yost et al., 1996) and activation of Wnt signaling (Larabell et al., 1997). Therefore, we overexpressed several mutant β-catenin constructs and examined the distribution of endogenous β-catenin in animal caps by confocal microscopy. Since our anti–β-catenin antibody recognizes the NH2-terminal domain (Yost et al., 1996), we used TM–β-catenin mutants that lack the NH2-terminal domain to distinguish endogenous β-catenin protein from ectopic β-catenin proteins. We found that overexpression of TM–β-catenin 1–9 resulted in the increased accumulation of endogenous β-catenin in nuclei (Fig. 4, A–F). Specifically, after injection of TM–β-catenin we observed that a subset of animal cap cells show elevated levels of endogenous β-catenin in nuclei (Fig. 4, A and D, arrowheads). Since a fluorescent lineage tracer (Oregon green dextran [OGDx]; Fig. 4, B and E) was coinjected with the RNA, simultaneous analysis of cells possessing elevated nuclear β-catenin and the lineage tracer (Fig. 4, C and F) reveals a direct correspondence between the presence of TM–β-catenin and accumulation of endogenous β-catenin in the nucleus. Neighboring cells not receiving TM–β-catenin 1–9 RNA do not show high levels of endogenous β-catenin in the nucleus (Fig. 4, A, C, D, and F, arrows). In addition, a small percentage of cells (∼10%) that received TM–β-catenin RNA also does not show high levels of endogenous β-catenin in the nucleus. The reason for this result is unclear, but it may simply represent differences in the responsiveness of individual cells to TM–β-catenin expression, variations in the levels of TM–β-catenin RNA received by each cell, or cell cycle differences in the levels of endogenous β-catenin in the nucleus. The effect of overexpression of TM–β-catenin on the localization of endogenous β-catenin was confirmed in four separate experiments in which ∼15 animal cap explants were analyzed. Identical results to those presented in Fig. 4 were also obtained with a TM–β-catenin ΔN mutant, which only lacks the amino-terminal domain (data not shown). In contrast to the effect of TM–β-catenin expression on nuclear β-catenin levels, injection of a control RNA encoding β-catenin ΔN-9 (Fig. 1 A), a mutant that lacks the NH2-terminal domain and Arm repeats 1–9 and is not active in the axis duplication assay (data not shown), does not affect levels of endogenous β-catenin in the nucleus (Fig. 4, G–I). Thus, ectopic expression of β-catenin causes an unsuspected accumulation of endogenous β-catenin in nuclei, similar to overexpression of activators of the Wnt pathway (Schneider et al., 1996; Yost et al., 1996; Larabell et al., 1997). The simplest explanation is that ectopic β-catenin present in the cytoplasm or even at membranes competes with endogenous β-catenin for access to the degradative machinery, resulting in the stabilization and accumulation of endogenous β-catenin in the nucleus, a hypothesis tested below.


Analysis of the signaling activities of localization mutants of beta-catenin during axis specification in Xenopus.

Miller JR, Moon RT - J. Cell Biol. (1997)

Overexpression of TM–β-catenin results in the accumulation of endogenous β-catenin in the nucleus. RNA encoding TM– β-catenin 1–9 (A–F) was coinjected with Oregon green dextran (OGDx; B, E, and H) as a lineage tracer to determine the effect of TM– β-catenin overexpression on the distribution of endogenous β-catenin (A, C, D, and F). Merged images (C and F) demonstrate that cells  overexpressing TM–β-catenin 1–9 possess high levels of endogenous β-catenin in nuclei (arrowheads) in contrast to that observed in  nonexpressing cells (arrows). Overexpression of ΔN-9 β-catenin (G–I), that does not promote axis duplication, does not affect the levels  of endogenous β-catenin in the nucleus (G and I; arrowheads mark nuclei of cells expressing the ΔN-9 mutant and arrows mark nuclei of  nonexpressing cells).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139814&req=5

Figure 4: Overexpression of TM–β-catenin results in the accumulation of endogenous β-catenin in the nucleus. RNA encoding TM– β-catenin 1–9 (A–F) was coinjected with Oregon green dextran (OGDx; B, E, and H) as a lineage tracer to determine the effect of TM– β-catenin overexpression on the distribution of endogenous β-catenin (A, C, D, and F). Merged images (C and F) demonstrate that cells overexpressing TM–β-catenin 1–9 possess high levels of endogenous β-catenin in nuclei (arrowheads) in contrast to that observed in nonexpressing cells (arrows). Overexpression of ΔN-9 β-catenin (G–I), that does not promote axis duplication, does not affect the levels of endogenous β-catenin in the nucleus (G and I; arrowheads mark nuclei of cells expressing the ΔN-9 mutant and arrows mark nuclei of nonexpressing cells).
Mentions: Given that the TM–β-catenin mutant appears to signal by elevating a free, signaling pool of endogenous β-catenin, we sought to determine whether overexpression of TM– β-catenin results in the accumulation of endogenous β-catenin in nuclei in a manner similar to that seen after inhibition of GSK-3 (Yost et al., 1996) and activation of Wnt signaling (Larabell et al., 1997). Therefore, we overexpressed several mutant β-catenin constructs and examined the distribution of endogenous β-catenin in animal caps by confocal microscopy. Since our anti–β-catenin antibody recognizes the NH2-terminal domain (Yost et al., 1996), we used TM–β-catenin mutants that lack the NH2-terminal domain to distinguish endogenous β-catenin protein from ectopic β-catenin proteins. We found that overexpression of TM–β-catenin 1–9 resulted in the increased accumulation of endogenous β-catenin in nuclei (Fig. 4, A–F). Specifically, after injection of TM–β-catenin we observed that a subset of animal cap cells show elevated levels of endogenous β-catenin in nuclei (Fig. 4, A and D, arrowheads). Since a fluorescent lineage tracer (Oregon green dextran [OGDx]; Fig. 4, B and E) was coinjected with the RNA, simultaneous analysis of cells possessing elevated nuclear β-catenin and the lineage tracer (Fig. 4, C and F) reveals a direct correspondence between the presence of TM–β-catenin and accumulation of endogenous β-catenin in the nucleus. Neighboring cells not receiving TM–β-catenin 1–9 RNA do not show high levels of endogenous β-catenin in the nucleus (Fig. 4, A, C, D, and F, arrows). In addition, a small percentage of cells (∼10%) that received TM–β-catenin RNA also does not show high levels of endogenous β-catenin in the nucleus. The reason for this result is unclear, but it may simply represent differences in the responsiveness of individual cells to TM–β-catenin expression, variations in the levels of TM–β-catenin RNA received by each cell, or cell cycle differences in the levels of endogenous β-catenin in the nucleus. The effect of overexpression of TM–β-catenin on the localization of endogenous β-catenin was confirmed in four separate experiments in which ∼15 animal cap explants were analyzed. Identical results to those presented in Fig. 4 were also obtained with a TM–β-catenin ΔN mutant, which only lacks the amino-terminal domain (data not shown). In contrast to the effect of TM–β-catenin expression on nuclear β-catenin levels, injection of a control RNA encoding β-catenin ΔN-9 (Fig. 1 A), a mutant that lacks the NH2-terminal domain and Arm repeats 1–9 and is not active in the axis duplication assay (data not shown), does not affect levels of endogenous β-catenin in the nucleus (Fig. 4, G–I). Thus, ectopic expression of β-catenin causes an unsuspected accumulation of endogenous β-catenin in nuclei, similar to overexpression of activators of the Wnt pathway (Schneider et al., 1996; Yost et al., 1996; Larabell et al., 1997). The simplest explanation is that ectopic β-catenin present in the cytoplasm or even at membranes competes with endogenous β-catenin for access to the degradative machinery, resulting in the stabilization and accumulation of endogenous β-catenin in the nucleus, a hypothesis tested below.

Bottom Line: Given this unexpected result, we focused on the membrane-tethered form of beta-catenin to resolve the apparent discrepancy between its membrane localization and the hypothesized role of nuclear beta-catenin in establishing dorsal cell fate.Compared with nonphosphorylated beta-catenin, beta-catenin phosphorylated by glycogen synthase kinase-3 preferentially associates with microsomal fractions expressing the cytoplasmic region of N-cadherin.These results suggest that protein-protein interactions of beta-catenin can be influenced by its state of phosphorylation, in addition to prior evidence that this phosphorylation modulates the stability of beta-catenin.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, University of Washington School of Medicine, Seattle 98195, USA.

ABSTRACT
In Xenopus embryos, beta-catenin has been shown to be both necessary and sufficient for the establishment of dorsal cell fates. This signaling activity is thought to depend on the binding of beta-catenin to members of the Lef/Tcf family of transcription factors and the regulation of gene expression by this complex. To test whether beta-catenin must accumulate in nuclei to establish dorsal cell fate, we constructed various localization mutants that restrict beta-catenin to either the plasma membrane, the cytosol, or the nucleus. When overexpressed in Xenopus embryos, the proteins localize as predicted, but surprisingly all forms induce an ectopic axis, indicative of inducing dorsal cell fates. Given this unexpected result, we focused on the membrane-tethered form of beta-catenin to resolve the apparent discrepancy between its membrane localization and the hypothesized role of nuclear beta-catenin in establishing dorsal cell fate. We demonstrate that overexpression of membrane-tethered beta-catenin elevates the level of free endogenous beta-catenin, which subsequently accumulates in nuclei. Consistent with the hypothesis that it is this pool of non-membrane-associated beta-catenin that signals in the presence of membrane-tethered beta-catenin, overexpression of cadherin, which binds free beta-catenin, blocks the axis-inducing activity of membrane- tethered beta-catenin. The mechanism by which ectopic membrane-tethered beta-catenin increases the level of endogenous beta-catenin likely involves competition for the adenomatous polyposis coli (APC) protein, which in other systems has been shown to play a role in degradation of beta-catenin. Consistent with this hypothesis, membrane-tethered beta-catenin coimmunoprecipitates with APC and relocalizes APC to the membrane in cells. Similar results are observed with ectopic plakoglobin, casting doubt on a normal role for plakoglobin in axis specification and indicating that ectopic proteins that interact with APC can artifactually elevate the level of endogenous beta-catenin, likely by interfering with its degradation. These results highlight the difficulty in interpreting the activity of an ectopic protein when it is assayed in a background containing the endogenous protein. We next investigated whether the ability of beta-catenin to interact with potential protein partners in the cell may normally be regulated by phosphorylation. Compared with nonphosphorylated beta-catenin, beta-catenin phosphorylated by glycogen synthase kinase-3 preferentially associates with microsomal fractions expressing the cytoplasmic region of N-cadherin. These results suggest that protein-protein interactions of beta-catenin can be influenced by its state of phosphorylation, in addition to prior evidence that this phosphorylation modulates the stability of beta-catenin.

Show MeSH
Related in: MedlinePlus